Investigating the role of CIZ1 in the maintenance of imprinted X-inactivation

Abstract

X-chromosome inactivation (XCI) is a way of dosage compensation that occurs in female eutherian mammals. There are two modes of XCI: Imprinted XCI where the paternal X is silenced predominantly, and random XCI where one of the two X chromosomes is randomly inactivated. Xist, a lncRNA expressed from the inactive X (Xi), spreads throughout Xi and recruits various factors that establish epigenetic modifications to transcriptionally silence X-linked genes. Xist interacts with several RNA Binding Proteins that play roles in Xist-mediated gene regulation. One of these proteins is the Cip1-interacting Zinc finger protein 1 (CIZ1), a nuclear matrix protein enriched at the Xi. CIZ1 colocalizes with the Xist RNA and ciz1 knockout leads to the disruption of Xist localization and loss of repressive marks. However, these functions are in the context of random XCI. In this project, we aimed to explore the possible role of CIZ1 in imprinted XCI. This study will provide insights into the evolutionary aspect of XCI and help in gaining knowledge about the conservation of mechanisms/pathways involved in the two types of XCI. We strategized to obtain CRISPR-Cas9 mediated knockout of ciz1 in XEN cells and study the Xist localization and X-linked gene expression. However, we observed the presence of CIZ1 despite a frameshift deletion. We are now working on shRNA mediated knockdown of CIZ1 to obtain maximal reduction of CIZ1 and investigate the dose dependence of CIZ1 in imprinted XCI, because the function of CIZ1 is dosage sensitive in random XCI. Currently, I am screening single-cell clones for CIZ1 knockdown.